Viral-mediated activation and inhibition of programmed cell death

Viruses are ubiquitous intracellular genetic parasites that heavily rely on the infected cell to complete their replication life cycle. This dependency on the host machinery forces viruses to modulate a variety of cellular processes including cell survival and cell death. Viruses are known to activate and block almost all types of programmed cell death (PCD) known so far. Modulating PCD in infected hosts has a variety of direct and indirect effects on viral pathogenesis and antiviral immunity. The mechanisms leading to apoptosis following virus infection is widely studied, but several modalities of PCD, including necroptosis, pyroptosis, ferroptosis, and paraptosis, are relatively understudied. In this review, we cover the mechanisms by which viruses activate and inhibit PCDs and suggest perspectives on how these affect viral pathogenesis and immunity.


Introduction
Programmed cell death (PCD) plays a fundamental role in organismal development, infectious disease pathogenesis, immune regulation, and tissue homeostasis. Dysregulated PCD is associated with a wide variety of diseases, including immunological and developmental disorders, neurodegeneration, and cancer. Hence, understanding the mechanisms of PCD regulation is essential to devise strategies to manipulate PCD to manage several disorders.
PCD is initiated after cells sense genotoxic, metabolic, and infectious insults. During virus infection, sensing of viral-derived nucleic acids can initiate signaling to activate PCD. To a varying degree, viruses utilize the infected cell transcription and translation machinery to complete their life cycle; thus, produce nucleic acid replication intermediates that are identified as non-self by a variety of host-encoded nucleic acid sensors [1]. Detection of nucleic acids activates a plethora of interconnected antiviral pathways including activation of the interferon (IFN)-mediated antiviral state, translational arrest, and PCD. In a concerted approach, these processes restrict virus propagation until the development of virus-specific adaptive immunity [1]. Recently, it has been shown that emission of immunomodulatory secretomes after PCD shapes the development of adaptive immunity. There are several elegant reviews that document the immunogenicity of PCD [2].
While the mechanisms by which pathogen-associated molecular pattern (PAMP) sensing activates antiviral state have been extensively studied, so much remains unknown about how  [5], and translational arrest that can directly or indirectly influence the induction of PCD [6]. In addition, several virus families encode proteins to inhibit the function of nucleic acid sensors [7], various antiviral factors such as IFN-stimulated genes (ISGs), and even proteins involved in the initiation and execution of PCD [8]. Hence, it is still unknown what types of nucleic acid receptors are directly involved in the induction of newly emerging PCD types. In this review, we provide literature on the mechanisms by which viruses activate and inhibit PCD and how that affects viral pathogenesis and host immunity.

Sensing of viral-associated nucleic acids
Availability, localization, and structure of viral-associated nucleic acids in infected cells provide host-encoded sensors to selectively identify non-self-nucleic acids. Viral-associated nucleic acids are sensed by cell surface-expressed (Toll-like receptor 3 (TLR3)), endosomelocalized (TLR3, TLR7, TLR8, TLR9), and cytoplasmic sensors (retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5)/nucleotide-binding oligomerization domain-like receptors (NLRPs) such as NLRP1 [9], NLRP6-DHX15 [10], NLRP9-DHX9 [11]/cyclic GMP-AMP synthetase (cGAS) stimulator of IFN genes (STING), absent in melanoma 2 (AIM2). Viral RNA is mainly recognized by TLR3 and RIG-I-like helicases (RLHs) (RIG-I/MDA5), whereas DNA is sensed by cGAS, AIM2-like receptors (ALRs), and TLR9. While most RNA sensors are well established, numerous DNA receptors continue to be discovered, including the human DNA sensor DNA-PK that functions independent of STING [12]. Species and cell-specific effects may account for the plethora of DNA sensors postulated, for instance, deletion of the ALR locus did not affect the IFN response to cytosolic DNA, dispelling the role of ALR in mice [13]. Most nucleic acid receptors, including the TLR family, the RLHs, and cGAS-STING, directly or indirectly induce transcription factors, including IFN-regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB), which up-regulate the expression of antiviral effector proteins, chemokines, and cytokines. RLHs and STING-cGAS are major nucleic acid sensors that activate phosphorylation-induced oligomerization of adaptor proteins mitochondrial antiviralsignaling (MAVS) and STING, respectively. Oligomerized MAVS and STING are scaffolding proteins closely associated with the IkB kinase (IKK)-related kinases TANK-binding kinase (TBK1) and IKK-e, which are responsible for phosphorylation of IRF3/7 [14-16], and IKK-b and IKK-a, which phosphorylate NF-kB [17]. IRF3/7 and NF-kB are transcription factors that bind regions in the promoter to up-regulate the expression of IFNs and proinflammatory cytokines, respectively [18,19].
Binding of secreted IFN-a/b to IFN receptors activates the associated Janus kinases (JAKs) tyrosine kinase 2 (TYK2) and JAK1, which phosphorylate both signal transducer and activator of transcription 1 (STAT1) and STAT2. STAT1, STAT2, and IRF9 form the IFN-stimulated gene factor 3 (ISGF3) complex that translocates to the nucleus to bind IFN-stimulated response elements (ISREs) and promote the expression of hundreds of ISGs. ISGs have a variety of functions including antiviral defense and initiation of adaptive immunity and collectively create a nonpermissive antiviral state [20].
Although not a focus of this review, there are also nucleic acid receptors with direct antiviral activity, including double-stranded RNA (dsRNA)-activated protein kinase R, 2 0 5 0 oligoadenylate synthetase 1 (OAS1) and adenosine deaminase acting on RNA 1 (ADAR1). OAS1 and ADAR1 recognize foreign nucleic acids to directly act on viral RNA by either inhibiting translation or degradation of the target RNA.

Mechanisms of programmed cell death activation after virus infection
In addition to the secretion of proteins with direct antiviral effect, nucleic acid sensing pathways involving RLHs, TLRs, Z-DNA/RNA binding protein 1 (ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI), NLRPs, and cGAS-STING play a role in the activation of apoptosis, necroptosis, and ferroptosis. Overall, the different types of PCD activated during virus infection involve distinct signaling pathways, some exclusively relying on caspases (apoptosis and pyroptosis) and others depending on kinases (necroptosis) and lipid peroxidation sensors (ferroptosis). In the following sections, we present a concise summary of how viruses activate the various PCD pathways.

Mechanisms of apoptosis induction during virus infection
Apoptosis is one of the most researched viral-induced PCD. In virus-infected cells, apoptosis can be initiated via the extrinsic or intrinsic pathway (Fig 1; [21]). In the extrinsic pathway, apoptosis begins after activation of the death domain receptors by the tumor necrosis factor (TNF) family of proteins including Fas activated by the FasL ligand, TNF-related apoptosisinducing ligand (TRAIL) receptors 1 and 2 activated by TRAIL, and TNF receptor 1 (TNF-R1) activated by TNF [21,22]. Ligand binding to Fas and TRAIL receptors leads to oligomerization of the death receptors and assembly of the death-inducing signaling complex (DISC) consisting of death receptor, the adaptor molecule FADD (FAS-associated with a death domain), procaspase-8, procaspase-10, and cellular FADD-like interleukin-1β (IL-1β)-converting enzyme (FLICE)-like inhibitory protein (c-FLIP). c-FLIP has 2 isoforms, long and short, which control the activation of caspase cascade that emanates from caspase-8-mediated apoptotic and nonapoptotic signaling. In contrast to Fas and TRAIL death receptors, TNF-R1-mediated apoptotic signaling is complex and recruits TNF-R-associated protein with death domain (TRADD) as an adaptor protein, which subsequently recruits FADD, TNF-associated factor-2 (TRAF-2), receptor-interacting protein (RIP), and RIP-associated IL-1β-converting enzyme homolog (ICH-1)/cell death protein-3 (CED-3)-homologous protein with a death domain (RAIDD). FADD then binds and activates the extrinsic apoptosis initiator caspase-8, which subsequently activates the effector caspases-3/6/7 [23].
In the intrinsic pathway, several proapoptotic stimuli result in mitochondrial outer membrane permeabilization [24,25]. Mitochondrial permeabilization permits the release of mitochondrial intermembrane space proteins, such as cytochrome c into the cytosol [24]. This series of events activates the intrinsic apoptosis initiator caspase-9 and downstream effector caspases that are the same as the extrinsic pathway [24]. The B-cell lymphoma 2 (BCL-2) family of proteins regulate the intrinsic pathway at the level of the mitochondria [26]. Some of the BCL-2 family of proteins such as BCL-2 and myeloid leukemia 1 (MCL-1) are antiapoptotic, while others including BCL-2-associated X protein (BAX), BCL-2 antagonist or killer (BAK), and BCL-2 homology domain (BH3)-interacting domain death agonist (BID) are proapoptotic [24]. The antiapoptotic proteins play a role to inhibit proapoptotic proteins BAX/BAK by sequestering BH3-only proteins such as BID [27].
Many viruses can induce apoptosis through one or both of the apoptosis pathways. . IRF3 is well characterized for its function as a transcription factor to stimulate the expression of IFN-mediated antiviral genes [35]. In some instances, IRF3 is also involved in the induction of apoptosis through a pathway commonly referred as the RLR-induced IRF3-mediated pathway of apoptosis (RIPA) [35]. Successful RIPA activation requires IRF3 to interact with BAX via its BH3 and to undergo translocation to the mitochondria, leading to the activation of apoptosis. This process requires additional proteins that are distinct from the antiviral transcription pathway [35]. Activation of the RIPA

Mechanisms of necroptosis induction during virus infection
Necroptosis, a regulated form of necrosis, is often activated after engagement of Toll-like receptors (TLRs), death receptors, and the intracellular sensors Z-DNA/RNA binding protein 1 (ZBP1)/DAI [60,61]. Necroptosis is caspase independent and relies on TNF and Z-DNA/ RNA to initiate the signaling cascade and kinases RIPK1/3 and mixed lineage kinase domainlike pseudokinase (MLKL) for execution (Fig 2). The morphological changes associated with necroptosis are distinct from apoptosis; these include organelle swelling and cell lysis [62]. In addition, during necroptosis, RIPK1/3 heterodimers form a complex that activates NF-kBmediated proinflammatory response to allow the emission of cytokines/chemokines [63-65]. Furthermore, damage-associated molecular patterns (DAMPs), such as ATP and HMGB1, are also profusely secreted during necroptosis [66,67].

Canonical (indirect) activation of necroptosis.
The initiation of necroptosis through TNF, also known as the indirect necrosome formation and canonical pathway, leads to the formation of TNF-receptor-associated complex, which independently recruits receptor-interacting protein I (RIPK1) and TRADD through their death domains (DDs) [

Mechanisms of pyroptosis induction during virus infection
Pyroptosis plays a crucial role in regulating host immunity against viruses. It is a form of inflammatory cell death that can be initiated after recognition of conserved PAMPs by host pathogen recognition receptors (PRRs) (Fig 3). The molecular response of host cells to pyroptosis is characterized by pore formation, plasma membrane rupture, and the activation and release of proinflammatory cytokines.
A critical step in pyroptosis activation is gasdermin cleavage [98]. Gasdermin proteins are cleaved by caspases into N-and C-terminal fragments. The N-terminal portion of gasdermin Overall, pyroptosis is initiated through 2 distinct pathways: the canonical pathway and the noncanonical pathway. The canonical pathway of pyroptosis, otherwise known as caspase-1-dependent PCD, is commonly activated by several viruses. In contrast, the noncanonical pathway that results in the activation of caspase-4, caspase-5, and caspase-11, after sensing of bacterial PAMPs, thus will not be discussed in this review.
A second signal is required for the pyrin domain of NLRP3 to bind ASC, causing ASC oligomerization and ASC speck formation [116]. The internal CARD and activation domain recruits and activates caspase-1 [117]. Caspase-1 activation leads to the downstream cleavage of N-terminal gasdermin and pore formation. The exact mechanism for the execution of the second signal is not known.
Two relatively new inflammasomes NLRP6 and NLRP9 can also activate pyroptosis. Activated NLRP6 along with ASC form an inflammasome to activate caspase-1 and caspase-11 [10,11,104]. Furthermore, NLRP9b is one of the 3 mouse paralogs of NLRP9 and recognizes short dsRNA stretches in concert with the RNA helicase Dhx9 to form inflammasome complexes with the adaptor protein ASC to activate caspase-1-mediated gasdermin D (Gsdmd)induced pyroptosis and the maturation of IL-18 [118-120]. . The CARD domain of procaspase-1 is recruited through the CARD domain of ASC, allowing for inflammasome assembly and caspase-1 activation. The exact mechanism by which viral DNA is exposed for AIM2 sensing is not precisely known, although several viral dsDNA ligands ofAU : Pleasenotethattheab VACV, human papillomavirus (HPV), hepatitis B virus, and mouse cytomegalovirus (MCMV) are sensed in an AIM2-dependent manner [124,125].

IFI16 inflammasome-mediated viral pyroptosis.
Closely associated with the AIM2 inflammasome is the IFI16 inflammasome, a canonical inflammasome with an ability to bind single and double-stranded DNA, triggering pyroptosis during viral infection. Initially, IFI16 was discovered as a viral DNA sensor of HSV-1, VACV, Kaposi sarcoma-associated herpesvirus (KSHV), and single-stranded DNA from HIV-infected CD4+ T cells [126]. IFI16 is mainly localized within the nucleus; however, it is also known to translocate to the cytoplasm to engage in antiviral immune response [126,127]. As a regulator of pyroptosis, cytoplasmic IFI16 can colocalize with ASC via its PYD domain after viral DNA recognition by the HIN domain [128]. Such interactions allow for caspase-1 activation, leading to cytokine release and pyroptosis.
During its latent period, KSHV, a dsDNA virus, resides within the nucleus of infected cells, tethered to host cell chromatin. During KSHV infection, oligomers of IFI16 can be seen to colocalize in the nucleus of infected endothelial cells with the KSHV genome in a nonspecific way; this cumulative interaction launches inflammasome formation, which recruits caspase-1 and ASC, inducing pyroptotic cell death and mature inflammatory cytokine release [129]. It is most probable that KSHV binding to IFI16 results in structural changes that allow its interaction with ASC [130].

Mechanisms of ferroptosis activation during virus infection
Ferroptosis is a type of PCD that is initiated after sensing free iron accumulation and increased lipid peroxidation. These two are coupled to or caused by the accumulation of intracellular reactive oxygen species (ROS) [131,132]. Lipid peroxidation refers to the result of oxidative attack on polyunsaturated fatty acids by ROS. One of the ways that ferroptosis is induced is via the inhibition of the system Xc − cystine/glutamate antiporter, which is a heterodimer made of solute carrier (SLC) family 7 member (SLC7A11) and SLC3A2. This antiporter functions to import cystine into the cell where it is reduced to cysteine and used for glutathione (GSH) synthesis [133]. GSH works to reduce ROS in the cell via the function of GSH peroxidases such as glutathione peroxidase 4 (GPX4). Many molecules that induce ferroptosis, such as erastin, induce ferroptosis by inhibiting system Xc − or modulating the pathway in some way [132]. Morphologically, ferroptosis mainly manifests as shrinkage of mitochondria with increased membrane density and reduction in or vanishing of mitochondrial cristae, which is a different process from other modes of cell death [132].
Studies elucidating the mechanisms of ferroptosis induction in virus-infected cells are very limited. One study demonstrated that Newcastle disease virus (NDV) can induce ferroptosis [134]. Cells infected by NDV demonstrated an increase in ROS as well as lipid peroxides. The mechanism by which NDV induces ferroptosis is by inhibiting the Xc-GSH-GPX4 pathway and appears to require the p53 protein [134]. Another study highlighted that the expression of the hepatitis A virus 3C protease induced ferroptosis [135]. There is a direct link between the DNA sensor STING and ferroptosis. The classic ferroptosis inducer erastin induces the accumulation of STING in the mitochondria to ROS production and lipid oxidation [136]. Both of these are hallmarks of ferroptosis, and depleting STING1 reduced sensitivity to ferroptosis [136]. In another study, zalcitabine-induced mitochondrial DNA stress activates STING-mediated DNA sensing pathway leading to autophagy dependent ferroptotic cell death via lipid peroxidation, independent of the IFN response [137]. These studies highlight the need for future studies to determine how other viruses activate and inhibit ferroptosis.

Mechanisms of paraptosis activation during virus infection
Paraptosis is a type of PCD characterized by cytoplasmic vacuolation, usually involving the swelling of the ER and mitochondria. Paraptosis is often accompanied by an alteration of Ca 2+ and redox homeostasis [138], as well as by accumulation of misfolded proteins causing ER stress and cell death [139]. However, these features are not always present in cells undergoing paraptosis. In most cases, paraptosis depends on mitogen-activated protein kinase (MAPK) family members, such as c-Jun N-terminal protein kinase 1 (JNK1), p38, and mitogen-activated protein kinase kinase 2 (MEK-2), and it can be inhibited by the multifunctional adapter protein AIP-1/Alix. Similar to ferroptosis, there is little research on virus-induced paraptosis. Zika virus infection can activate ER-dependent, large, cytoplasmic vacuoles characteristic of paraptosis. The ability of the virus to create these paraptotic vacuoles was dependent on its ability to replicate within ER-derived membranes [140]. In another study, Singapore grouper iridovirus (SGIV) induced STAT-3-dependent paraptosis of fish cells [141]. Further research is required to understand mechanisms of virus-induced paraptosis.

Inhibition of programmed cell death by viruses
Depending on the type of virus, infection cells attempt to abort the infection by activating PCD. On the other hand, viruses highjack a variety of cellular processes including the initiation and execution of PCD starting early after infection. For example, herpesviruses and poxviruses, which utilize over one-third of their genome to counteract the host antiviral response [142]. In this section, we summarize the various strategies viruses deploy to inhibit apoptosis, necroptosis, and pyroptosis (Tables 1 and 2).
Viruses can also block apoptosis by inhibiting a variety of caspases. Expressed during cowpox virus (CPXV) infection, CrmA was the first serpin-like protein found to potently inhibit caspase-

Viral-mediated inhibition of necroptosis
Viruses inhibit necroptosis mostly at the level of RIPK1/3 and MLKL. In herpesviruses and poxviruses, inhibiting the kinases RIPK1/3 is a very common process. Both viruses encode various inhibitors of RHIM-dependent functions of RIPK1/3. Multiple herpesviruses encode  Poxviruses also target RIPK3 through viral inducers of RIPK3 degradation (vIRD). vIRD analogs are found in CPXV, variola virus (VARV), monkeypox virus (MPXV), and Ectromelia virus (ECTV) and directly bind to RIPK3 through their Ankyrin repeats, while their Fbox domains link to host SKP1-Cullin1-F-box machinery. This interaction stimulates ubiquitination of RIPK3 and subsequent proteasomal degradation [176]. Interestingly, VACV, an attenuated virus used to eradicate VARV, lacks a functional vIRD homolog potentially shedding light on its benign and immunogenic nature [176].
In summary, the large diversity and redundancies in viral inhibition of necroptosis support the emerging theory that necroptosis and viruses have coevolved specific virus-host mechanisms to antagonize the other, further highlighting importance of necroptosis in antiviral immunity and viral replication.

Viral-mediated inhibition of pyroptosis
Overall, the mechanisms by which viruses inhibit pyroptosis remain largely unknown. However, plus sense RNA viruses, such as Coronaviridae, utilize viral proteases to inhibit pyroptosis PLOS PATHOGENS [182][183][184] (Table 2). Highly conserved viral proteases, such as NSP5 in coronaviruses, directly bind and cleave Gasdermin D in porcine and human cells, producing a truncated N-terminal Gasdermin D, lacking essential residues from caspase-1 cleavage [98]. Interestingly, a singular large dsDNA virus, African swine fever virus, has shown a similar mechanism through the viral protease S273R [185].

Perspectives and future studies
The complexity of viral sensing pathways, diversity of animal viruses, and the ability of viruses to modulate PCD using their accessory proteins has been challenging to build a universal framework to dissect how viral ligand sensing interacts with the PCD machinery. Most studies we reviewed use live virus infections to study PCD. Hence, it is largely unknown how viral genome sensing in the context of inactivated and replication-defective virus stimulation regulates the PCD machinery. Many viral nucleic acid-like ligands including synthetic dsRNA: Poly I:C [193] and cGAMP [194] can activate cell death, suggesting the likelihood that viral genomes may also directly activate PCD, although similar studies are limited. In relation to this, it remains unknown if viral RNA and DNA genomes engage distinct PCD pathways.
An outcome of nucleic acid sensing is establishment of antiviral state via expression of various ISGs that exert direct antiviral effects in many ways. Although some ISGs have been extensively studied [195], the modes of action of a large majority of ISGs are still unknown [196]. There are more than 15 putative ISGs with proapoptotic functions, although their direct effect on PCD regulation is not known [197][198][199]. Furthermore, a large-scale proteomics study identified more than 200 novel PCD-associated proteins directly interact with some of the 104 inducibly expressed ISGs [196]. In support of this, IFN-induced transmembrane proteins 3 (IFITM3) directly regulates PCD induction in Zika virus-infected cells [140,200] and overexpression of ISG54 [201] and ISG12b2 [202], independent of IFN stimulation, activate apoptosis. The broader relevance of ISG modulating PCDs in the context of viral infection remains to be investigated.
Much is unknown how PCD influences viral pathogenesis. On one side, activation of PCD in an infected cell can curb virus replication, while some necrotic forms of PCD can assist spreading of virus to the surrounding tissue. In addition, certain PCDs can facilitate the identification of viruses by the adaptive immunity via emission of immunomodulatory secretomes that enhance antigen presentation [203]. To the infected host, activation of certain inflammatory PCDs may exacerbate disease pathogenesis, as these forms of PCD may enhance acute cytokine storms and/or potentially trigger delayed immunopathologies such as autoimmune disorders. Studies investigating how viral induced PCDs may instigate host reactivity against self are currently lacking.